Fig. 2.
PAK3 kinase phosphorylates GluA1-S863 in heterologous cells, in vitro, and in neurons. (A) GluA1 was immunoprecipitated (IP) from HEK cells following coexpression with an empty vector, constitutively active (CA) Rac1, Cdc42, or RhoA either alone or in conjunction with Myc-PAK3 or HA-MRCK (as indicated at Top). Lysates were prepared and processed for Western blot (WB) analyses using the indicated antibodies (Right). (B) Pharmacological inhibition of PAK disrupts GluA1-S863 phosphorylation. Cortical neurons cultured 18 d in vitro were pretreated for 1 h with vehicle (DMSO) or 10 μM PAK inhibitor before 20 min application of 1 μM TTX. Lysates were prepared for immunoprecipitation of endogenous GluA1, followed by Western blot analyses (as indicated at Right). (C) GluA1 and either PAK Type I (Myc-PAK1 and Myc-PAK3) or Type II (Myc-PAK6) family members were coexpressed HEK cells in conjunction with Cdc42 CA or Rac CA. Lysates prepared for immunoprecipitation of GluA1(IP) and total extract aliquots (INPUT) were analyzed by Western blot with α-S863(P), total GluA1, Myc, and endogenous Cdc42 or Rac1 antibodies (indicated at Right). (D) Purified PAK3 phosphorylates GluA1 in vitro. Immunoblots using α-S863(P) (Top) or GST antibody (Middle) and autoradiography (Bottom) of purified GST and GST‐GluA1 C-terminal used in kinase assays in the presence (+) or absence (−) of active PAK3. (E and F) Loss of endogenous PAK3 from cortical neurons abrogates TTX-mediated enhancement of GluA1-S863 phosphorylation. (E) Rat cortical neurons transfected by electroporation at DIV 0 with vector control (pSuper) or shRNA directed against either rat PAK1 (riPAK1) or rat PAK3 (riPAK3) remained untreated (control) or were stimulated with 1 μM TTX for 20 min following 18 d in vitro. Lysates were prepared for immunoprecipitation of endogenous GluA1, followed by Western blot analyses using α-S863(P) (Left) or total GluA1 (Right) antibodies (as indicated at Right). (F) Quantification of TTX stimulated versus unstimulated fold increases in S863(P) levels normalized to total immunoprecipitated GluA1. Error bars indicate ± SEM. *p < 0.03, ANOVA. n =11.